The disclosure invention relates to insecticidal toxins produced by or isolated from bacteria, especially Xenorhabdus, and methods for the use of toxin-containing compositions or isolated toxins as insecticides and/or as insect control agents, and which exhibit antimicrobial activity, as well.
Biological agents are an important option for management of insect pests. One method of insect control explored was the use of certain genera of nematodes. Nematodes, including the Steinernema and Heterorhabditis genera, might be useful as biological agents, in part because of their transmissible insecticidal bacterial symbionts of the genera Xenorhabdus and Photorhabdus. Upon entry into the insect, the nematodes release their bacterial symbionts into the insect hemolymph where the bacteria reproduce and eventually cause insect death. The nematode then develops and reproduces within the insect cadaver. Bacteria-containing nematode progeny exit the insect cadaver as infective juveniles, which can then invade additional larvae to repeat the cycle of insect death and nematode propagation. While this cycle is easily performed on a micro scale in a laboratory setting, adaptation to the macro level, as needed to be effective as a general use insecticide, is difficult, expensive, and inefficient.
There are now also pesticide control agents commercially available that are naturally derived. These naturally derived approaches can be as effective as synthetic chemical approaches. One such naturally occurring agent is the crystal protein toxin produced by Bacillus thuringiensis (Bt). These Bt protein toxins have been formulated as sprayable insect control agents. Another application of Bt technology is the genetic modification of plants to contain and express the genes that encode the Bt toxins for protection from plant pests. Another microbial insecticide is the spinosyn complex of polyketide molecules produced by the bacterium Saccharopolyspora spinosa. Spinosyn has been formulated as a spray for insect pests including house flies and mosquitoes. See, e.g., Kirst, H. A. (2010) J. Antibiotics 63:101-111; Sheehan et al. (2006) J. Nat. Prod. 69:1702-1710.
The genus Xenorhabdus is a member of the family Enterobacteriaceae; Xenorhabdus occurs in nature in a symbiotic association with the entomopathogenic nematode Steinernema. The nematode is the vector for transmission of the bacteria between insects. The bacteria produce a complex of protein toxins that kill the insect victim. The nematode reproduces in the insect cadaver ultimately differentiating to an infective juvenile stage, each carrying Xenorhabdus cells in its intestine. Another related bacterium, Photorhabdus, is similarly associated with the nematode Heterorhabditis (Mol. Microbiol. 64: 260. 2007. Ann Rev Microbiol. 63; 557. 2009).
Xenorhabdus and Photorhabdus both produce antibiotic molecules that are thought to maintain the insect cadaver as an efficient growth chamber for their respective nematode associates (Current Opinions Chemical Biol. 13: 224. 2009. Appl. Environ. Microbiol. 61:4329. 1995), and both produce toxins that are toxic when injected or fed to insects. This was first discovered for Photorhabdus by Bowen and Ensign (Appl Environ Microbiol. 64: 3029. 1998). A complex of four proteins varying from 30 to 200 kDa, named the tc complex, was shown to be toxic when fed to a wide variety of insect pests including mosquito larvae (WO97/17432. 1997).
Subsequently, a variety of insecticidal proteins produced by one Xenorhabdus species, X. nematophila, were described. Morgan et al (Appl. Env. Microbiol. 67, 2062. 2001) isolated several species of X. nematophila that produced a 280 kDa protein complex that was orally active against Pieris brassiceae (cabbage moth) larvae. Brown et al (J. Biol. Chem. 279:14595. 2004) reported a 42 kDa toxin lethal when injected into two species of insect larvae. Ribeiro et al described a 10,790 Da protein that lyses insect hemocyte cells and mammalian red blood cells, however activity against insects was not reported. Vigneux et al (J. Biol. Chem. 282: 9571. 2007) reported similar cell damaging activity caused by a 78 kDa protein produced by the same species of bacteria, but again insect toxicity was not reported. Khandelwal et al (Appl. Env. Microbiol. 69: 2032. 2003) reported the insecticidal activity of a large molecular complex of proteins associated with outer membrane vesicles of X. nematophilus and later these authors reported a 17 kDa protein isolated from the complex that is orally toxic to Helicoverpa armigera larvae and cytotoxic to hemocytes of the insect (J. Bacteriol. 2004:6465. 2004). Lang et al (J. Nat. Prod. 71: 1074. 2008) isolated three low molecular weight peptides from X. nematophila that are insecticidal, and one of the peptides, 662 Da in size, showed weak insecticidal activity against one insect and against a crustacean (brine shrimp, Artemia). A family of five lysine rich cyclopeptide antibiotics, none with insecticidal activity, was described by Gualtieri et al (J. Antibiotics 62:295, 2009). Significantly, there are limited reports of mosquitocidal toxins produced by Xenorhabdus bacteria. U.S. Pat. Nos. 6,048,838 and 6,379,946 report mosquitocidal activity for proteins produced by twelve natural isolates of Xenorhabdus. 
The sequenced genome of Photorhabdus luminescens tto1 revealed two genes coding for proteins which, when produced in recombinant Escherichia coli, were toxic to larvae of three mosquito species (Duchaud et al Nature Biotech 21:1307. 2003). The gene products were shown to be proteins of 45 and 14 Da (Waterfield et al FEMS Microbiol Lett 245:47. 2005). The same toxin, named PirAB, produced by another Photorhabdus species, P. asymbiotica, was lethal to Aedes aegypti and A. albopictus mosquitoes (Appl. Env. Microbiol 75: 4627. 2009). US WO 97/17432 (Ensign et al.) reported mosquitocidal activity for the tca complex of insecticidal proteins produced by P. luminescens strain W14. The toxin XLT described in the present application is believed to be the first low molecular weight mosquitocidal lipopeptide of either Xenorhabdus or Photorhabdus. 
Bacteria of the genus Xenorhabdus are symbiotically associated with the Steinernema nematode. These bacteria only had pesticidal activity when injected into insect larvae and did not exhibit biological activity when delivered orally (see Jarosz J. et al. Entomophaga 36 (3) 1991 361-368; Balcerzak, Malgorzata. Acta Parasitologica Polonica, 1991, 36(4), 175-181).
There are Xenorhabdus toxins which are produced and secreted by growing bacterial cells of the genus Xenorhabdus. The protein complexes, with a native molecular size ranging from about 800 to 3000 Da, can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins exhibit significant toxicity upon exposure to a number of insects. Furthermore, toxin activity can be modified by altering media conditions. In addition, the toxins have characteristics of being proteinaceous, with activity that is heat labile and sensitive to proteolysis. See U.S. Pat. No. 6,048,838, for example.
Because mosquitoes are a common insect pest and are also important as vectors of serious human disease, it is important to find environmentally friendly, economical and effective means for their control.
It has been difficult to effectively exploit the insecticidal properties of the nematode or its bacterial symbiont. Thus, it would be quite desirable to discover insecticidal agents derived from Xenorhabdus bacteria that have oral activity so that the products produced therefrom could either be formulated as a sprayable insecticide or the bacterial genes encoding said proteinaceous agents could be isolated and used in the production of transgenic plants.
There is a need in the art for environmentally friendly, economical and effective means of insect control, especially mosquito control for benefits in human and animal health and the comfort of both humans and animals.